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as a : Taxonomy and Chemistry

Monique McHenry, Ph.D. Assistant Professor of , University of Vermont Co-founder, CASE Institute Disclaimer & Disclosure

• I am a member of the faculty at the University of Vermont • Materials presented here represent my own findings, views and opinions and should not be taken as a statement, position, opinion, or endorsement by the University of Vermont

University of Vermont Health Network

Discussion includes investigational not approved for use in the .

DISCLOSURE: Is there anything to disclose? Yes Please list the Potential Conflict of Interest (if applicable): Relationships with Vermont Patients Alliance and Phytoscience Institute All Potential Conflicts of Interest have been resolved prior to the start of this program. Yes (If no, credit will not be awarded for this activity.) All recommendations involving clinical medicine made during this talk were based on evidence that is accepted within the profession of medicine as adequate justification for their indications and contraindications in the care of patients. Yes Cannabis history

Human use 12,000-10,000 years ago begins 1753 Linnaeus classifies L. 2011 First Cannabis genome is sequenced

Policy 1930 Harry Anslinger began the process of criminalization and including marijuana and by 1931 29 states had outlawed Cannabis. 1970 Controlled Substances Act passed. Cannabis is Schedule one categorized to have a high potential for abuse and no medicinal value. 1996 Passage of the Compassionate Use Act initiative

Medical discovery 1964 (THC) is isolated and synthesized 1988 Cannabinoid receptors CB1 and CB2 discovered 1992 Endogenous cannabinoids discovered

3 Cannabis history

Hemp is removed from CSA

10,000 BCE - 0 Used as a Gains popularity as a Criminalized therapeutic drug therapeutic drug Cannabis history

Source: By Lokal_Profil, CC BY-SA 2.5, https://commons.wikimedia.org/w/index.php?curid=2370050 Cannabis history

• The ancestral distribution of Cannabis is central Asia (Li, 1974)

• Cannabis arrived in the Americas after 1545 (Small et al., 1975)

• Currently Cannabis is distributed world-wide

Photo credits: Hillig and Mahlberg, 2004; Angiosperm Phylogeny Group, 2015 Cannabis plant biology

Macroscopic morphological characters – Leaves, flowers, and

Photo credit: http://en.wikipedia.org/wiki/Cannabis Cannabis plant biology

Microscopic morphological characters - Trichomes and glands Cannabis plant biology

Reproductive characters

– Male and female flowers are found on different Female flower Male flower

– Wind Pollinated

Pollen

Photo credit: http://plant.pictures.com; http://www.internationalhempassociation.org/jiha/jiha5208.html Cannabis plant biology

Plants produce chemical compounds, phytochemicals, as part of their normal metabolic activities. Phytochemicals are divided into two groups:

1. Primary metabolites • found in all plants, serve basic plant functions • i.e. sugars and fats

2. Secondary metabolites • found in select plants, serve in a Wounded , the more specific function fruit exuding opium active principle of • i.e. opium, phytocannabinoids opium

Slide content contributed by Dr. Wolfgang Dostmann, UVM LCOM Cannabis plant biology

Plants produce chemical compounds, phytochemicals, as part of their normal metabolic activities. Phytochemicals are divided into two groups:

1. Primary metabolites • found in all plants, serve basic plant functions • i.e. sugars and fats

2. Secondary metabolites Cannabis glandular Delta-9- • found in select plants, serve in a trichomes containing tetrahydrocannabinol, more specific function phytocannabinoids one active principle of • i.e. opium, phytocannabinoids Cannabis

Slide content contributed by Dr. Wolfgang Dostmann, UVM LCOM Cannabis chemicals

Slide content contributed by Dr. Wolfgang Dostmann, UVM COM; source Potter, 2009 Cannabis chemicals The main biologically active phytocannabinoids

Delta-9-tetrahydrocannabinol (THC) • Partial Agonist CB1/CB2 receptors • Psychoactive • Anti-, anti-nausea • Anti-spasm, anti-immune

Cannabidiol (CBD) • Antagonist CB1/CB2 receptors • Not psychoactive • Anti-seizure activity

Activated by heating the plant Slide content contributed by Dr. Wolfgang Dostmann, UVM LCOM Cannabis chemicals

What are Phytocannabinoids?

Slide content contributed by Dr. Wolfgang Dostmann, UVM LCOM Cannabis chemicals How are Phytocannabinoids manufactured by the plant?

Phytocannabinoid biosynthesis

1. Chemical precursors are absorbed through the roots from the soil

2. CBD & THC derive from CBG compounds, catalyzed by the enzyme THCA synthase or CBDA synthase • CBDA synthase is dominant • Hemp has THCA & CBA synthase • Drug-type (Marijuana) has THCA & CBDA synthase • nonfunctional CBDA synthase Source: Weilben et al., 2015 Cannabis chemicals

Phytocannabinoids evolved in Cannabis

Herbivory defense? Environmental response? Cannabis chemicals

Slide content contributed by Dr. Wolfgang Dostmann, UVM LCOM Cannabis chemicals

What are the non-cannabinoids?

Terpenes (Myrcene) Slide content contributed by Dr. Wolfgang Dostmann, UVM LCOM Cannabis chemicals

Myrcene has an effect

“Terpenes such as myrcene may constitute a lead for the development of new peripheral with a profile of action different from that of the -like drugs.”

Slide content contributed by Dr. Wolfgang Dostmann, UVM LCOM Cannabis chemicals

α-Pinene appears to be a broad-spectrum antibiotic

•Does α-Pinene in three hemp cultivars have an inhibitory activity of microbial growth? •Yes, results demonstrate α-Pinene (in hemp essential oils) significantly inhibits microbial growth • Gram (+ and -) bacteria • Yeasts •Conclusion: industrial hemp essential oils applications could control spoilage, food-borne pathogens, and phytopathogen microorganisms.

Slide content contributed by Dr. Wolfgang Dostmann, UVM LCOM Cannabis chemicals

Boiling point Partition Coefficient, logP Myrcene 166 °C (330 °F) 4.30

Limonene 176 °C (349 °F) 3.40

Linalool 198 °C (388 °F) 2.65 α-Pinene 155 °C (311 °F) 2.80 Geraniol 230 °C (446 °F) 3.28

THC 157 °C (315 °F) 6.97 CBD 180 °C (356 °F) 6.33 CBN 185 °C (365 °F) 6.93

Slide content contributed by Dr. Wolfgang Dostmann, UVM LCOM Cannabis chemicals Why does Cannabis produce Phytocannabinoids?

Level of Phytocannabinoids Cannabis chemicals Why does Cannabis produce Phytocannabinoids? What we know….

Plant organ matters!

Highest amount of THC & CBD is found in flower (Andre et al., 2016) Cannabis chemicals Why does Cannabis produce Phytocannabinoids? What we know….

Size matters! The amount of THC is correlated to the size of resin gland (Small and Naraine, 2015) Cannabis chemicals Why does Cannabis produce Phytocannabinoids? What we know…. Light quality matters!

•Magagnini et al. (2018) Phytocannabinoid yield changes with different light sources –Perhaps complex mechanisms mediated by the UV-A and blue wavelengths that induce CBG accumulation (CBG is precursor of other phytocannabinoids) g flower/plant % THC % CBD

• Lydon et al. (1987) In “drug-type” plants, increasing levels of UV-B Cannabinoid/g radiation corresponded with increased levels of THC –Potential for selective pressure to favor “drug-type” plants in environments with high levels of UV radiation Cannabis chemicals Why does Cannabis produce Phytocannabinoids? Desiccation Prevention?

•Major Phytocannabinoids increased when intentional drought applied seven weeks into reproductive stage (Caplan, 2018) Caplan, 2018 Hot and Cool and •Wild plants in cool humid environments dry humid had fewer glandular hair compared to plants in hot dry environments (Sharma, 1975)

•Higher THC levels measured in drier continental climate compared to maritime climate (Murari et al., 1983)

Sharma, 1975 Cannabis chemicals Why does Cannabis produce Phytocannabinoids? Stress?

• Correlation between low plant height and high Phytocannabinoid content • Low Mg, higher THC & CBD content (Coffman and Gentner, 1975) Cannabis chemicals Why does Cannabis produce Phytocannabinoids?

Insecticide? • THC-rich Mexican (vs. CBD-rich Turkish) Cannabis was reported fatal to tiger moth (Arctia caja) larvae (Rothschild et al., 1977) Cannabis chemicals Why does Cannabis produce Phytocannabinoids? Chemical plant defense?

• Cannabis extract inhibited germination of Lettuce (Lactuca sativa) (Mahmoodzadeh et al., 2015)

• Cannabis extract decreased the germination of monocots (Pedulko, 2014) Cannabis chemicals Why does Cannabis produce Phytocannabinoids?

Disease prevention?

– Harbor antibiotic properties (ElSohly et al. 1982)

– Antibiotic properties assist in the overwintering of seed (Ferency, 1956) Cannabis chemicals Why are the amounts of Phytocannabinoids inconsistent?

Plant age changes Phytocannabinoid content

• THC increases throughout the life cycle of Cannabis before reaching a plateau (De backer et al., 2012)

https://www.cannabisreports.com/news/2016/03/07/study-examines- cannabinoid-terpene-development-patterns-three-cannabis- chemotypes/ Cannabis chemicals Why are the amounts of Phytocannabinoids inconsistent?

Phytocannabinoid difference due to changes in:

• Genetics • Plant Organ • Light • Soil composition • Available moisture • Age

Aizpurua-Olaizola et al., 2016 Cannabis chemicals Is it possible to grow Cannabis with reproducible terpene and Phytocannabinoid levels? • Yes! As long as environmental conditions and genetics are standardized – Fischedick et al. (2010) • 11 “strains” • 36 compounds • Control for grow cycle time, plant stress, different genotypes Cannabis chemicals

How is Phytocannabinoid and terpene production related? • Levels of cannabinoids and terpenoids correspond

(Fischedick et al., 2010) Cannabis chemical research

Are there variations in the chemical composition of Cannabis grown under Title 18 of the Vermont Statutes?

35% % CBD % THC % CBN

30%

25%

20%

15%

10%

5%

0% 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Cannabis chemical research

Are there variations in the chemical composition of Cannabis grown under the Industrial Hemp Program in Vermont? Parameters controlled: • Organ harvested • Light • Soil composition • Available moisture • Harvest Age

Variables: • Genetic composition Cannabis chemical research

Are there variations in the chemical composition of Cannabis grown in different environmental conditions under Title 18 of the Vermont Statutes? Cannabis chemical research

Are there variations in the chemical composition of Cannabis grown in different environmental conditions under the Industrial Hemp Program in Vermont?

Parameters controlled: • Organ harvested • Genetic composition • Soil composition • Available moisture • Harvest Age

Variables: • Light Cannabis chemical research Does Cannabis grown with different nutrients yield different chemical compositions?

• Different fertilization concentrations produced changes in vegetative growth, biomass yield, and phytocannabinoid concentrations

• Increasing fertilizer rate had a dilution effect on THC, THCA, and CBGA, despite increased biomass yield (Caplan et al., 2017; doi: 10.21273/HORTSCI12401-17). A1. Indica vegeta ve-growth curve for 5 B1. Primary outcome of yield, measured as dried- C 1. Secondary outcome of yield, measured as total different po ng soil condi ons (ver cal height flower weight in grams/plant, for Indica above-ground biomass (wet weight in grams/plant), for over me). morphotypes. Indica morphotypes. 140 120 GB5 GB1 100 GB4 GB2 80 GB3 GB3 60 GB2 GB4 40 GB1 GB5 20 Cannabis chemical research 0 200 400 600 800 1000 0 A2. Hybrid vegeta ve-growth curve for 5 A1. Indica vegeta ve-growth curve for 5 DoesB2 .Cannabis Primary outgrowncome o fwith yield ,different measured anutrientss dried- yield Cdifferent2. Second achemicalry outcome compositions? of yield, measured as Bto1t. aPlr imary outcome of yield, measured as dried- C 1. Secondary outcome of yield, measured as total different po ng soil condi ons (ver cal height differenat bpooven-g rsoouiln cdo nbdiiomonass (sv (ewr ecta wl heeiigghht in grams/ fplolawnetr) ,w feoirg ht in grams/plant, for Indica above-ground biomass (wet weight in grams/plant), for flower weight in grams/ plant, for hybrid morphotypoevse. r me). over me). Dried flower weight Hybrid morphotypes. morphotypes. Indica morphotypes. 160 140 140 Treatments 120 GB5 PH1 PH5 GB1 120 1 100 GB4 PH2 GB2 100 2 PH4 80 GB3 Parameters controlled: GB3 80 PH3 60 PH3 3 GB2 • Organ harvested GB4 60 PH4 40 4 • Light PH2 GB1 40 GB5 PH5 • Available moisture 20 20 5 PH1 0 200 400 600 800 1000 A1. Indica vegeta ve-growth curve for 5 B1. Primary outcome of yield, measured as dried- C •1. SHarvestecondar yAge outcome of yield, measured as to0ta l different po ng soil condi ons (ver cal height 0 A2. Hyb0ri d vege2ta00v e-gr4ow00th cur6ve0 0 f or 5 800 1000 flower weight in grams/plant, for Indica ab•oveGenetic-ground composition biomass (wet weight in grams/planBt2)., Pforimr ary outcome of yield, measured as dried- C2. Secondary outcome of yield, measured as total over me). different po ng soil condi ons (ver cal height A3. Sa va vegeta ve-growth curve for 5 morpPercenthotype Phytocannabinoids. by weight Indica morphotypes. flower weight in grams/ plant, for hybrid morphotypes. above-ground biomass (wet weight in grams/ plant), for Vegetativeover me) growth. curve different po ng soil condi ons (ver cal height 140 B3. Primary outcome of yield, measured as dried- C3. Secondary outcome of yield, measured a1s6 0t otal Hybrid morphotypes. 14 Treatments Variables: over me). flower weight in grams/ plant, for Sa va morphotypes. above-ground biomass (wet weighTreatmentst in grams/ plant), for 120 1 140 GB5 12 GB1 • SSoila v acomposition morphotypes 1 PH1 PH5 160 120 100 2 GB4 14100 GB2 2 100 PH2 PH4 80 SD 1 SD 5 120 3 80 GB3 PH3 8 GB3 3 PH3 60 SD 2 SD 4 100 60 GB2 PH4 6 G4 B4 PH2 40 80 SD 3 4 40 SD 3 PH5 4 5 GB1 PH1 60 GB5 5 20 20 SD 4 SD 2 402 0 200 400 600 800 1000 0 0 200 400 600 800 1000 0 SD 5A 3. Sa va vegeta ve-growth curve for 5 SD 1 200 A2. Hybrid vegeta ve-growth curve for 5 difCfe2r.e Snet cpoondnagr syo oil ucotcnodmi oen os f( vyeirelcda,l mheeigahstu red as toBta3l. Primary outcome of yield, measured as dried- C3. Secondary outcome of yield, measured as total B2. Primary outcome of yield, measured as dried- flower weight in grams/ plant, for Sa va morphotypes. above-ground biomass (wet weight in grams/ plant), for different po ng soil condi ons (ver cal height 0 ovaebr ovme0e- )g. r oun2d00 b iom4a0s0s (we6t 0w0e ight8 i0n0 g ram10s0/ 0p lant), for flower weight in grams/ plant, for hybrid morphotypes. Sa va morphotypes over me). 160 160 Hybrid morphotypes. 140 140 SD 1 SD 5 PH1 120 PH5 SD 2 120 100 SD 4 PH2 100 80P H4 SD 3 SD 3 PH3 60 80 PH3 SD 4 SD 2 60 40 PH4 PH2 SD 5 SD 1 40 20 PH5 0 0 200 400 600 800 1000 20 PH1 0 0 200 400 600 800 1000 A3. Sa va vegeta ve-growth curve for 5 different po ng soil condi ons (ver cal height B3. Primary outcome of yield, measured as dried- C3. Secondary outcome of yield, measured as total over me). flower weight in grams/ plant, for Sa va morphotypes. above-ground biomass (wet weight in grams/ plant), for Sa va morphotypes 160 140 SD 1 SD 5 120 SD 2 100 SD 4 80 SD 3 SD 3 60 SD 4 SD 2 40 SD 5 20 SD 1 0 0 200 400 600 800 1000 Cannabis: the name

Taxonomy • Family: Cannabaceae • Genus: Cannabis • Species? – At least 13 Cannabis species have been described

Why is the scientific name important? Cannabis: the name Why worry about using a scientific name? Consistency! “hemp” usually = C. sativa, but the term has been applied to many fiber crops • Manila hemp = Musa textilis (banana anyone?) • Sisal hemp = Agave sisalana (related to tequila!) • Sunn hemp = Crotolaria juncea (pea family) Cannabis: the name What is the difference between hemp and marijuana?

Chemical content!

< 0.3% THC content = Hemp

• Plants can generate different chemical constituents due to the environment in which they grow • The different chemical compositions within the same plant species are called chemical phenotypes, or “chemotypes” Cannabis: the name Is there a genetic difference between hemp and marijuana? Cannabis genome and transcriptome – Published 2011 – Sequenced DNA and RNA with Illumina Next-generation technology – SNP identified differences in “hemp” and “marijuana genome

Van Bakel et al. (2011) Cannabis: the name Is there a genetic difference between hemp and marijuana?

Phylogenetic neighbor network of ~3000 SNP alignment from the Cannabis genome Lynch et al., 2015 Cannabis: the name Is there a chemical difference between hemp and marijuana?

Yes, if chemotypes are based on CBD/THC ratio • Hillig and Mahlberg (2004) suggest distinct groups Cannabis: the name Is there a chemical difference between hemp and marijuana?

Yes, if chemotypes are based on avgerage percentage of major Phytocannabinoid mass for dried and un-pollenated female flowers (Lynch et al., 2015) Cannabis: the name Cannabis: the name Are the drug-type names Cannabis sativa and viable? No, Cannabis indica should not be used • Plants grouped morphologically as C. indica (red) or C. sativa (blue) do not form distinct groups based on chemical composition • one species concept Cannabis sativa L.

Elzinga et al. (2015) Cannabis: the name

Do drug-type Cannabis “strains” have different chemotypes?

No, “strains” do not have not consistently reproducible chemical compositions

Elzinga et al. (2015) Cannabis names and chemicals What’s Next?

Source: http://galaxy.phylosbioscience.com/?source=website Cannabis names and chemicals What’s Next?

International Code of Nomenclature for Cultivated Plants

• Established in 1953 • Rules • Name is unique • Follows principle of priority • Operates within the Botanical code (International Code of Nomenclature for algae, fungi, and plants ) • Does not regulate trademarks or marketing names • trade designations • Cannabis “strains” should be identified as cultivars once stabilized

Why does Cannabis impact humans?

Cannabis impacts humans through the endocannabinoid (eCB) system.

The eCB system consists of: • cannabinoid receptors (CBs) • endogenous agonists • agonist-metabolizing enzymes

Slide content contributed by Dr. John McPartland, UVM LCOM Why does Cannabis impact humans?

• CB1 is primarily expressed in neurons • CB1 is found in adipose tissue, blood vessels, gut, testes, uterus • CB1 is not found in the brainstem’s cardiorespiratory drive Autoradiograph of rat brain exposed to centers – which explains [3H]CP55,940 (Herkenham et al., 1990). = highest densities in memory the lack of lethal overdoses centers, limbic system, basal from cannabis ganglia, cerebellum  Unlike receptor = lower densities in cerebral cortex distribution = lowest densities in the brain stem

CB2 is primarily expressed in the immune system Slide content contributed by Dr. John McPartland, UVM LCOM Cannabis clinical research

Methods • compilation of data from randomized clinical trials (RCTs) comparing cannabinoids to placebo for many conditions • information about potential for adverse events (AEs) with cannabis

Results • Improvement in pain when compared to placebo overall (Odds Ration 1.41 [0.99-2.00]) • Cannabinoids were associated with approximately 3x increased odds of any AE compared to placebo Slide content contributed by Dr. Kalev Freeman, UVM LCOM Cannabis clinical research

FDA Approved: Synthetic THC

• Dronabinol (Marinol™) for Pain or Nausea

• Nabilone (Cesamet™) for Pain or Nausea

FDA Approved: CBD

• (Epidiolex®) Cannabis clinical research

• Risk of pediatric exposure • Risk of diversion for recreational use • No known risk of death from the drug itself • Margin of exposure (MOE) is the ratio between toxicological threshold (benchmark lethal dose) and estimated human intake based on individual and population scenarios • MOE analysis shows that by a wide margin, cannabis is the least risky recreational drug (Lachenmeiera DW and Rehm J., 2015)

Slide content contributed by Dr. Kalev Freeman, UVM LCOM Cannabis pre-clinical research

Cannabidiol (CBD) activates non-endocannabinoid receptors • TRPV1 is found in nervous tissue

• TRPV1 is involved in the feeling of pain

• TRPV1 activity can be decreased with overactivity = desensitization • Can lead to analgesic effects

Iannotti, F.A. et al., 2014; doi: 10.1021/cn5000524 Cannabis clinical research

Why not just pharmaceutical products? 1. Dronabinol not well tolerated, Epidiolex just became available, and Sativex is not yet approved in USA 2. When approved, costs could be exorbitant 3. “Whole plant” or “Full spectrum” extracts have other cannibinoids and terpenes with effects on CB receptors

Are plant-based products more effective (and less expensive) than pharmaceuticals?

Can they be provided in a safe and consistent fashion? Slide content contributed by Dr. Kalev Freeman, UVM LCOM Cannabis research Label accuracy on CBD products Cannabis research Label accuracy on CBD products Cannabis research Label accuracy on CBD

• All products contain CBD!

• Only one product had levels above 0.3% THC

• 50% of products were labeled accurately

• 17% of Vermont products were labeled accurately Questions?